1. Field of the Invention
The field of this invention is nerve cell growth regulation.
2. Background
The failure of the adult central nervous system (CNS) to regenerate after injury is a major clinical problem, affecting some 200,000 people in the United States alone. Despite intensive research, an effective approach in promoting significant regeneration of CNS nerve fibers remains lacking. The inability of CNS to regenerate is partly due to inhibitory factors associated with myelin, a cellular structure surrounding the nerve fibers.
There are currently few effective methods that can promote significant nerve regeneration of severed or damaged nerve fibers. A number of endogenous molecules are known to modulate neural cell growth (1). These factors may exert either attractive or repulsive action on the extension of axonal growth cones (1,2). Experiments in mammals have shown that blocking of some of the inhibitory factors by antibodies could promote regeneration of severed axons in the spinal cord and lead to functional recovery of limb movements.
Recent reports have shown that attractive responses to netrins, mediated by the DCC/UNC-40 family of proteins, can be converted to repulsion by coexpression of proteins of the UNC-5 family (3). In addition, attractive effects of brain-derived neurotrophic factor (BDNF) and netrin-1 on Xenopus spinal neurites in culture can be converted to repulsion by inhibition of protein kinase A activity (4,5). We disclose here the opposite phenomenon: that by specific pharmacological manipulations, the action of neural cell inhibitory (or repulsive) factors can be reversed. Such pharmacological treatments change the inhibitory nature of those inhibitory factors into supportive factors and thus promote nerve regeneration.
We have found that the action of many protein factors that either inhibit or promote nerve growth are mediated through two general mechanisms. Factors such as neurotrophins including brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) as well as certain netrins can promote nerve growth in vitro. Nerve fibers grow towards these factors when presented as a localized source. On the other hand, factors such as Semaphorin III, myelin-associated glycoprotein (MAG) and purified myelin, cause collapse of the nerve terminal and block nerve growth. When these factors present as a localized source, nerve fibers grow away from these factors. We have further found that the inhibitory or repulsive effects of the latter factors can be reversed by pharmacological treatments that activate cyclic nucleotide dependent protein kinases, e.g. by increasing the level of cyclic nucleotides, cAMP and cGMP. In addition, we found the action of protein factors that promote nerve growth, e.g. BDNF and NT-3, can also be enhanced by these cyclic nucleotides. Thus by pharmacological interventions that elevate the cyclic nucleotide levels, we are able to help nerve regeneration associated with injuries of the nervous system.
Accordingly, the invention provides methods and compositions for promoting neural cell growth and/or regeneration. The general methods involve contacting with an activator of a cyclic nucleotide dependent protein kinase a neural cell subject to growth repulsion mediated by a neural cell growth repulsion factor. The activator may comprise a direct or indirect activator of the protein kinase, including cyclic nucleotide analogs, activators of a cyclic nucleotide cyclase, NO inducers, inhibitors of a cyclic nucleotide phosphodiesterase, etc. The repulsion factor typically comprises one or more natural, endogenous proteins mediating localized repulsion or inhibition of neural cell growth. Examples include neural cell guidance proteins such as semaphorins, CNS myelin fractions or components thereof such as MAG, etc. The target cells are generally vertebrate neurons, typically injured mammalian neurons in situ. The subject compositions include mixtures comprising a neural cell, an activator of a cyclic nucleotide dependent protein kinase and a neural cell growth repulsion factor.